Transposase and method of gene modification

ABSTRACT

A transposase encoded by the Tol2 element; a polynucleotide encoding the same; a method of modifying the gene structure of a cell (preferably a vertebrate cell) by using the above protein; a method of modifying the function of a cell by modifying the gene structure thereof; and a cell having been modified in function by these methods. Also the structure of a cis element necessary in gene transfer is clarified and presented.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/148,639, filed Jun. 3, 2002 now U.S. Pat. No. 7,034,115, which isa U.S. national phase application under 35 U.S.C. § 371 of InternationalApplication Ser. No. PCT/JP00/08014, filed Nov. 14, 2000, which claimsthe benefit of Japanese Application Serial No. 2000-109033, filed Apr.11, 2000, and Japanese Application Serial No. 11-345508/1999, filed Dec.3, 1999. The entire contents of all of the above-referenced applicationsare incorporated herein by this reference.

TECHNICAL FIELD

The present invention relates to a new protein having a transposase-likeactivity, a transposase composed of the above protein, a method ofmodifying the gene structure of a cellular gene by using these proteinand transposase, a method of modifying the function of a cell by thismethod, a method of introducing a gene by this method, a plasmid usedfor this method, and a cell having been modified in function by thismethod.

BACKGROUND ART

The medaka fish (Oryzias latipes) is a teleost inhabiting in East Asiaand has been used for studying vertebrate genetics. The mutations at thei locus of the medaka fish cause amelanotic skin and red-colored eyes.This i locus is known to encode a gene for tyrosinase. From one of the ialleles, i⁴, DNA of about 4.7-kb was cloned and found to have atransposon-like sequence; that is to say, it included open readingframes homologous to transposases of transposons of the hAT familyincluding hobo of Drosophila, Ac of maize and Tam3 of snapdragon, andshort terminal inverted repeats. This medaka element was named Tol2. Thelaboratory strains of the medaka fish contain about 10 copies of thiselement per haploid genome.

In the i⁴ mutant fish, the Tol2 element found in the tyrosinase genelocus has been shown by PCR to be excised from the target locus duringembryonic development (Koga et al., 1996).

Zebrafish (Danio rerio), as well as the medaka fish (Oryzias latipes),is a small teleost and has been developed as a model animal to studyvertebrate genetics and development (Takeuchi, 1966; Yamamoto, 1967;Streisinger et al., 1981). In zebrafish, large-scale chemicalmutagenesis screens have been performed (Driever et al., 1996; Haffteret al., 1996), and, to facilitate cloning of the mutated genes, aninsertional mutagenesis method using a pseudotyped retrovirus has beendeveloped and performed (Lin et al., 1994; Gaiano et al., 1996;Amsterdam et al., 1997). Also, in an attempt to develop transposontechnologies that would allow enhancer trap and gene trap screens to beperformed, transposition of transposons of the Tcl/mariner family infish has been tested and demonstrated (Ivics et al., 1997; Raz et al.,1997; Fadool et al., 1998). Although these results are encouraging,neither highly efficient transgenesis nor insertional mutagenesismethods using a transposon have not yet been developed.

The present inventors have been interested in developing noveltransposon technologies using the Tol2 element. As a first step towardsthis goal, the present inventors developed a transient embryonicexcision assay using zebrafish embryos, in which zebrafish fertilizedeggs were injected with a plasmid DNA harboring the Tol2 element, showedthat the Tol2 element was excisable from the injected plasmid DNA, andindicated that the Tol2 element is an autonomous member and is active inzebrafish (Kawakami et al., (1998) Gene 225, 17-22). Although the DNAsequence of the Tol2 element is similar to those of transposases oftransposons of the hAT family, neither an active enzyme, which canfunction in trans, nor cis-elements essential for the excision reactionhave been identified. In order to develop the Tol2 element as a usefultool for transgenesis and insertional mutagenesis, it is necessary todissect and characterize cis and trans requirements. The functionaltransposase encoded by the Tol2 element had not yet been identifiedprior to the present invention.

DISCLOSURE OF THE INVENTION

The present invention first aims to identify mRNA transcribed from theTol2 element injected in zebrafish embryos. Secondly, in order todetermine whether the transcript encodes an active enzyme or not, thepresent invention develops a novel assay method, in which zebrafishfertilized eggs are co-injected with RNA synthesized in vitro using theTol2 cDNA as a template and a plasmid DNA harboring a nonautonomous Tol2element, which has a deletion in the transposase coding region.

The present invention also identifies the active trans-factor andessential cis-elements, that function in excision of the Tol2 element inzebrafish.

Consequently, the present invention results in a new protein encoded bythe Tol2 element and a polynucleotide encoding the same. Also thepresent invention, by using the above protein, results in a method ofmodifying the gene structure of a cell, preferably the gene structure ofa vertebrate, in a method of modifying the function of a cell bymodifying the gene structure thereof, and in a cell having been modifiedin function by these methods. Furthermore, the present inventiondiscloses the cis-elment structures essential for transposition, andpresents the same.

The present invention relates to a protein having the transposase-likeactivity, which has an amino acid sequence shown in SEQ ID NO:2, anoptionally substituted amino acid sequence with any replacements ordeletions in part of the original amino acid sequence, or an optionallysubstituted amino acid sequence with addition of other amino acids tothe original amino acid sequence. Also the present invention relates toa transposase comprising the said protein.

Further, the present invention relates to the nucleic acid encoding thesaid protein, wherein the nucleic acid is preferably DNA having anucleotide sequence shown in SEQ ID NO:1 or DNA which can hybridize tothe said DNA, or is the corresponding RNA.

The present invention reveals that the said protein has atransposase-like activity which catalyzes transposition of the abovetransposon, and relates to a method of modifying the gene structurecomprises the excision in part of a gene in a cell, preferably avertebrate cell, or the insertion of the excised part into any otherlocus in the presence of the said protein or the nucleic acid which canproduce the said protein. It is preferable that the said excised genehas nucleotide sequences containing at least one inverted repeat (theAngel elements ) in forepart of its nucleotide sequence.

Further, the present invention relates to a method of inserting aforeign gene into a gene of a cell, and a method of modifying a functionof a cell based on gene expression, and furthermore relates to a cellhaving been modified in function by the said method.

Also, the present invention relates to a plasmid used in these methodsand, more in detail, a plasmid which contains DNA having a nucleotidesequence that includes at least one inverted repeat sequence in theforepart of its nucleotide sequence.

Furthermore, in a method of inserting any DNA into the genomic DNA of avertebrate, the present invention relates to a method of inserting anyDNA into the genomic DNA of a vertebrate which is characterized byoperating the said insertion of DNA autonomously using the transposaseactivity, wherein a preferable DNA is the Tol2 element and thevertebrate is fish.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the Tol2 plasmid and the transcript, andthe structure of cDNAs described in the present invention. The dottedlines indicate introns. The inverted repeat (the Angel elements) in thefirst intron and positions of primers used in the present invention areshown by arrows.

FIG. 2 shows a comparison of amino acid sequences of transposases of theTol2 element described in the present invention (residues 106-335 of SEQID NO:2) and the Ac element (SEQ ID NO:3).

FIG. 3 shows a scheme for the transient embryonic excision assay byco-injection described in the present invention. Primers (tyr-ex4f andtyr-ex5r) used to detect the excision products are shown by arrows.

FIG. 4 shows photos, substitutes for drawings, which show the results ofthe PCR analysis of the excision reaction in zebrafish embryos describedin the present invention (SEQ ID NOs:4-9, respectively in order ofappearance).

FIG. 5 shows the structures of the (Tol2-tyr)ΔRV plasmid used fortransposition of the Tol2 element into the genome, the Tol2-tyr plasmidand Tol2 cDNA. The black line in the upper part of FIG. 5 shows theprobe used for Southern blot analysis.

FIG. 6 shows photos, substitutes for drawings, which show the results ofSouthern blot analysis of F1 progeny fish from each parental fish (ff-1and ff-7), wherein the presence of Tol2 is identified (FIG. 6, A) andshow the results of PCR (FIG. 6, B).

FIG. 7 shows the nucleotide sequences surrounding the Tol2 elementinserted in the genome of F1 progeny A (left: SEQ ID NO:10; right: SEQID NO:11), B (left: SEQ ID NO:12; right: SEQ ID NO:13) and C (left: SEQID NO:14; right: SEQ ID NO: 15) from ff-7.

BEST MODE FOR CARRYING OUT THE INVENTION

Previously, the present inventors injected the Tol2-tyr plasmid, aplasmid harboring the Tol2 element cloned from the tyrosinase genelocus, into zebrafish fertilized eggs and showed that the Tol2 elementis excisable from the injected plasmid DNA (Kawakami et al., 1998). Inorder to identify a transcript encoding a putative transposase activity,total RNA from embryos injected with the Tol2-tyr plasmid were prepared.The present inventors first performed 3′ RACE using four pairs of nestedprimers that annealed different parts of the Tol2 sequence.

Nested forward primers used to perform 3′ RACE are:

Tol2f2; 5′-TTGGTCAGACATGTTCATTG-3′ (SEQ ID NO:16) and Tol2f3;5′-ATGTTCATTGGTCCTTTGGA-3′, (SEQ ID NO:17) Tol2f4;5′-ATAGCTGAAGCTGCTCTGATC-3′ (SEQ ID NO:18) and Tol2f5; 5′-CTGCTCTGATCATGAAACAG-3′, (SEQ ID NO:19) Tol2f8; 5′-GCTTAATAAAGAAATATCGGCC-3′ (SEQID NO:20) and Tol2f9; 5′-AATATCGGCCTTCAAAAGTTCG-3′, (SEQ ID NO:21) andTol2f12; 5′-CTGTAATCAGAGAGTGTATGTGTA-3′ (SEQ ID NO:22) and Tol2f13;5′-ATTGTTACATTTATTGCATACAAT-3′. (SEQ ID NO:23)

cDNAs with polyadenylation were successfully amplified by 3′ RACE usingTol2f8 and Tol2f9, and Tol2f4 and Tol2f5, but not by 3′ RACE usingTol2f2 and Tol2f3, and Tol2fl2 and Tol2fl3.

Then, using nested reverse primers designed to perform 5′ RACE, Tol2r4;5′-CTCAATATGCTTCCTTAGG -3′ (SEQ ID NO:24) and Tol2r5;5′-CTTCCTTAGGTTTGATGGCG-3′ (SEQ ID NO:25), 5′ RACE was performed and thefull-length Tol2 transcript of 2156 nucleotides was identified (FIG. 1).

The cDNA sequence obtained is shown in SEQ ID NO:1.

FIG. 1 shows the structures of the Tol2 plasmids and its transcript. Thetop line of FIG. 1 shows the full-length Tol2 (Tol2-tyr). Dotted linesin the figure show introns. The inverted repeat (the Angel elements) inthe first intron and positions of the said primers are shown by arrows.The lower three lines of FIG. 1 show the results of 3′ RACE and 5′ RACE.In each case, introns are shown as dotted lines.

The fifth line shows the structure of the full-length mRNA. Thetranslated region corresponds to the nucleotide sequence between the85th (ATG) and the 2032nd nucleotide (TAG) of cDNA of sequence number 1.

The two lines in the bottom show the structures of deletion mutants,(Tol2-tyr)ΔRV, and (Tol2-tyr) Δin1ΔRV.

In the 5′ RACE analysis, aberrant transcripts that started from theplasmid sequence and jumped into cryptic splice acceptor sites in thefirst exon of the Tol2 element were also found (data not shown). Thesetranscripts were not studied further.

DNA sequencing of the cDNA revealed the exon-intron structure of theTol2 element (i.e., four exons and three introns) (as shown in the upperpart of FIG. 1). The cDNA encodes a protein of 649 amino acids. Theamino acid sequence of this protein is shown in SEQ ID NO:2.

Although the Tol2 element had been known to have a transposon-likesequence, the present invention for the first time identified that theTol2 element encodes a protein and the expression of the proteindescribed here generates the function. That is to say, the presentinvention results in a new protein encoded by the Tol2 element and alsoa polynucleotide which encodes the protein described here.

FIG. 2 shows a comparison of amino acid sequences of the proteindescribed in the present invention and a known transposase of atransposon of the hAT family. This comparison shows these proteins aresimilar, especially in the middle part (FIG. 2). But the amino acidsequences of NH₂— and COOH-terminus rather varies.

In order to determine whether the protein (the Tol2 transcript)identified in the present invention encodes a functional enzyme, a newtransient embryonic excision assay by co-injection was developed and, byusing this method, identification of the enzymatic activity wasperformed.

Zebrafish fertilized eggs were co-injected with mRNA synthesized invitro using the cDNA shown in the sequence number 1 as a template andthe (Tol2-tyr)ΔRV plasmid containing (Tol2-tyr)ΔRV (see FIG. 1), whichhas a deletion of the nucleotides between the EcoRV sites of the Tol2element. About 8 hours after the co-injection, DNA was prepared fromeach embryo and analyzed by PCR using primers,

-   tyr-ex4 f and tyr-ex5r,-   tyr-ex4f: 5′- GCTACTACATGGTGCCATTCCT-3′ (SEQ ID NO:26)-   tyr-ex5r: 5′-CACTGCCAGATCTGCTGGGCTT-3′ (SEQ ID NO:27)    which were prepared based on the sequence adjacent to the Tol2    element. FIG. 3 shows a scheme of this method and these primers are    shown in FIG. 4A.

PCR products of about 250 bp, indicative of excision of the Tol2 elementfrom (Tol2-tyr)ΔRV plasmid, was amplified in all embryos analyzed (56out of 56, see FIG. 4B lanes 1-10). This PCR product was never detectedfrom embryos injected only with the (Tol2-tyr)ΔRV plasmid DNA (0 out ofmore than 50, see FIG. 4B lanes 11-20).

The PCR products from six different embryos were cloned and sequenced.Three of them had the wild-type medaka fish tyrosinase gene sequence(FIG. 4C, excision product a), indicating that precise excision hadoccurred, and the other three had nearly wild type sequences withaddition of a few nucleotides (FIG. 4C, excision products b and c),characteristic to excision of transposons of the hAT family (Pohlman etal., 1984; Sutton et al., 1984; Koga et al., 1996; Kawakami et al.,1998), suggesting that the excision event in this experiment iscatalyzed by a transposase-like activity.

These results, i.e., when co-injected with the mRNA which has thenucleotide sequence shown in sequence number 1 of the present invention,the PCR product characteristic to excision of the transposon wasobtained and, when co-injected without the mRNA, such PCR products werenot obtained, indicate that the protein (the Tol2 transcript) describedin the present invention encodes a functional transposase, which cancatalyze the excision. Further, these results show that the(Tol2-tyr)ΔRV plasmid contains sequences of cis-elements essential forthe excision.

FIG. 4 shows the results of this experiment and arrows in FIG. 4A showpositions and directions of the primers used in the analyses. The upperpanel of FIG. 4B is a photo, a substitute for a drawing, which shows thePCR products using primers tyr-ex4f and tyr-ex5r, and the lower panelshows the PCR products using primers Tol2f1 and Tol2r3. In lanes 1-10,zebrafish embryos were injected with both the (Tol2-tyr)ΔRV plasmid andthe Tol2 mRNA, in lanes 11-20 the (Tol2-tyr)ΔRV plasmid alone wasinjected, and in lane G and P PCR products were amplified from 50 ng ofthe zebrafish genomic DNA and from 10 pg of the (Tol2-tyr)ΔRV plasmidDNA. FIG. 4C shows the DNA sequences of the excision products obtainedin the above experiments. The Tol2 sequence is shown in bold and 8 bpdirect repeat sequences flanking to the Tol2 element are underlined.

It was noted that, although the excision products could be detectedafter a single-round PCR amplification in the experiments in the presentinvention, two rounds of PCR were required in the previous analysis, inwhich fertilized eggs were injected with a sole plasmid DNA containingthe full-length Tol2 element without mRNA prepared in vitro. The higherefficiencies of the excision reaction observed here can be explained asmore transposases were supplied by RNA injection than those supplied byDNA injection.

The first intron of the Tol2 element contains about 300 bp of largeinverted repeats, and the repeat was recently identified as the Angelelement (Izsvak et al., 1999) (see FIG. 1). To test whether the sequencein the intron is essential for excision, we constructed the (Tol2-tyr)Δin1ΔRV plasmid, containing (Tol2-tyr) Δin1ΔRV (see the bottom of FIG.1), which completely lacked the sequences of the first intron, and itsactivity was analyzed by co-injection with the Tol2 mRNA as the saidmethod. This result is shown in FIG. 4D.

The upper panel of FIG. 4D is a photo, a substitute for a drawing, whichshows PCR products amplified using primers tyr-ex4f and tyr-ex5r and,the lower panel shows PCR products amplified using primers Tol2f1 andTol2r3. In lanes 1-8, zebrafish embryos were injected with both the(Tol2-tyr) Δin1ΔRV plasmid and the Tol2 mRNA, in lanes 9-12 both the(Tol2-tyr)ΔRV plasmid and the Tol2 mRNA were injected, and, in lanes13-16, the (Tol2-tyr) Δin1ΔRV plasmid alone was injected. Lane P showsthe PCR product amplified from 10 pg of the (Tol2-tyr) Δin1ΔRV plasmidDNA.

In lanes 9-12, the said experiment was conducted as controls and the PCRproducts indicating the excision could be detected, but the excisionproduct could not be detected in lanes 1-8 when the plasmid lacking theintron part (0 out of 16, see FIG. 4D lanes 1-8) was used, suggestingthat the first intron contains cis-elements essential for excision.

Further, the (Tol2-tyr) Δin1 plasmid, containing (Tol2-tyr) Δinl whichrestored theΔRV deletion and was about the same size as the (Tol2-tyr)ΔRV plasmid, i.e., which has a deletion between the 644th and 2163rdnucleotides of the Tol2 element, was also examined by the co-injectionassay, but PCR product indicating the excision could not be obtained (0out of 16, data not shown).

Although, further analyses using smaller deletions and point mutationsin the first intron sequence will be needed to define the essentialcis-sequences for excision precisely, since these results show that theintron part is essential for the excision and the intron contains theAngel elments as inverted repeats, it can be thought that the invertedrepeats are essential sequences for the excision described in thepresent invention.

Thus, we successfully identified for the first time the transcript (theprotein described in the present invention) encoded by the Tol2 elementand also identified a transposase activity of this protein andcis-sequences essential for transposition. These discoveries will leadto a biochemical characterization of the Tol2 transposase.

On the other hand, transposition of transposons belonging to theTcl/mariner family into the zebrafish genome has been reported (Raz etal., 1997; Fadool et al., 1998). In the experiments described in theirreports, zebrafish one-cell-stage embryos were co-injected withtransposase RNA transcribed in vitro and transposon vectors containingessential cis-sequences.

While a transposon belonging to a different family may have differentspecificities and efficiencies for insertion into the genome, by amethod of present invention which is a novel transposon technology infish using the Tol2 element, since its transposon excision procedure hasbeen carried out in the way of Raz et al., it might be possible totranspose DNA such as the Tol2 element into the genome in the wayconducted by using transposons of the Tcl/mariner family.

Therefore, we tested whether the Tol2 element can be inserted into thezebrafish genome by transposition. It is known that the zebrafish genomedoes not contain the Tol2 element.

To test whether the Tol2 element encodes a transposase that can catalyzetransposition, zebrafish fertilized eggs were co-injected with RNAtranscribed in vitro using the Tol2 cDNA as a template, which encoded aputative transposase, and a plasmid DNA harboring the (Tol2-tyr)ΔRVelement, which has a deletion in part of the region presumed to code thetransposase.

The structures of (Tol2-tyr)ΔRV plasmid and Tol2 cDNA are shown in FIG.5. 3′ and 5′ indicate the direction of transcription.

The injected eggs were raised to adulthood and mated to non-injectedfish. And the progeny fish were analyzed for the presence of the Tol2sequence.

Two out of eight injected fish could transmit the Tol2 sequence to theirprogeny. These two fishes were named ff-1 (founder fish-1) and ff-7(founder fish-7).

Two fish out of 68 F1 fish from the ff-1 fish had the Tol2 sequence.These two fish had the sequence of the plasmid portion as well as theTol2 sequence. On the other hand, 25 fish out of 50 F1 fish from theff-7 fish had the Tol2 sequence. These 25 fish did not have the plasmidsequence and were classified into three groups, A, B and C, from theresult of Southern blot shown in FIG. 6A. 7 fish were grouped as A, 3fish as B, and 15 fish as C.

FIG. 6A is a photo, a substitute for a drawing, which shows the resultof Southern blot analysis using a probe shown in FIG. 5, in which DNAsamples prepared from caudal fins of F1 fish from ff-1 and ff-7 weredigested with EcoRV. Two samples from ff-1 showed the same pattern butsamples from ff-7 showed three patterns, A, B and C.

Then, PCR analyses of F1 fish from ff-1 and ff-7 were performed. Primersused were shown in FIG. 5 as PCR1, PCR2 and PCR3. As controls, zebrafishgenomic DNA (G) and genomic DNA plus (Tol2-tyr)ΔRV plasmid DNA (G+P)were used. In F1 fish from ff-7, PCR products using PCR2 and PCR3 couldnot be amplified. This indicated that progeny fish from ff-7, unlikeprogeny fish from ff-1, did not have the plasmid sequence flanking tothe Tol2 element.

From the ff-7 progeny fish, DNA fragments containing the Tol2 sequenceand the flanking region were cloned by inverse PCR and sequenced. Ineach three case, A, B and C, the Tol2 sequence was surrounded byzebrafish genomic sequences and 8 bp duplications were created adjacentto the insertion. 8 bp duplications at both ends of the Tol2 element arecharacteristic to integration of transposons of the hAT family,indicating that the integration described here was catalyzed by atransposase.

FIG. 7 shows the determined nucleotide sequences of three types, A, Band C. Tol2 in FIG. 7 shows the Tol2 sequence. In A repeats of┌CTCAACTG┘, in B repeats of ┌TATAGAGA┘, and in C repeats of ┌GTTTTCAG┘were created at both ends of and adjacent to the Tol2 sequence.

In the vertebrate cultured cells and the germ line, transpositionactivities of Sleeping Beauty which was reconstituted and activatedartificially (Ivics, Z., et al., Cell, 91, 501-510 (1977)), Tc3 of C.elegans ( Raz, E., et al., Current Biology, 8, 82-88 (1977)) and marinerof Drosophila (Fadool, J.M., et al., Proc. Natl. Acad. Sci. USA, 95,5182-5186(1988)), all belonging to the Tcl/mariner family, have beenreported. No autonomous transposon activity residing endogenously in anyvertebrate genome, however, has been reported.

The present invention is the first report that identified an autonomouselement from a vertebrate genome and also for the first time reported afunctional transposase activity in vertebrate.

Therefore, the present invention relates not only to a method to excisea gene autonomously in vertebrate but also to a method to insert theexcised gene into any locus or any gene on the genome.

The protein in the present invention has the amino acid sequence shownin SEQ ID NO:2, but all of the amino acids shown there are notnecessarily required, and the protein in the present invention caninclude a protein having the transposase activity described in thepresent invention or similar activities described above (both of theseare called transposase-like activities) and also can include a proteinhaving replacement or deletion in part of amino acids of the aboveprotein, or having addition of any other amino acids to the aboveprotein. And preferably it has the amino acid sequence derived from theTol2 element. Further, the protein in the present invention includes aprotein which is produced from mRNA having the nucleotide sequencecorresponding to SEQ ID NO:1.

The nucleic acid in the present invention encodes the amino acidsequence which is related to the said protein, and preferably which hasthe polynucleotide having the sequence shown in SEQ ID NO:1. The nucleicacid in the present invention includes not only the said nucleotidesequence but also a nucleotide sequence which can hybridize to the saidnucleic acid, preferably under stringent conditions.

As for a method of modifying the gene structure of a gene in a cell inthe presence of the protein in the present invention or the nucleic acidwhich can produce the said protein, by introducing the protein or thenucleic acid, for instance the mRNA which can produce the proteindescribed here, and, at the same time, by introducing genes including agene to be transposed, for instance a plasmid, the gene structure in acell can be modified by the enzymatic activity of the protein in thepresent invention. The modification in the present invention is involvedpreferably in autonomous transposition. The cell is preferably an animalcell, more preferably a vertebrate cell, and much more preferably fishcell including a zebrafish cell.

Genes containing the said gene to be transposed can be substances whichdo not exist in a natural cell, such as a plasmid carrying a foreigngene to be transposed, and also can be a genomic gene existing in anatural cell. In this case, cis-elements required for transpositioncould be added to the gene if necessary. The gene to be transposed ispreferably a transposon, in certain circumstances, it may be a gene thatinsert a normal gene into the cell which has a disease caused byabnormalities of genes of various kinds.

Further, a method of modification in the present invention may onlyinclude the excision of part of a gene in a cell such as an insertedplasmid, however, may also include the insertion of all or part of thegene excised by this method, into any gene.

A gene excised in a method of modification in the present inventionpreferably has nucleotide sequences containing at least one invertedrepeat in the forepart of its nucleotide sequence. The inverted repeatis thought as a cis-elment or part of cis-elments for transposition ofthe gene.

Further, the present invention, by using the said methods ofmodification, relates to a method of introducing a foreign gene into agene of a cell and to a method of modifying a function of a cell basedon expression of the gene. By performing the said methods, for example,it is possible for a foreign gene on a plasmid to be transposed into thegenome in a cell, and for a new gene, which the cell concerned does notcontain originally, to be inserted into a cell. Further, by expressionof the newly inserted gene, it is possible to modify a function of acell. Furthermore, the present invention can result in a cell, whosefunction has been modified by this method. The said cell is preferableas a cell described in this method.

As a plasmid in the present invention which contains the nucleotidesequence having at least one inverted repeat in the forepart of itsnucleotide sequence, an optional substitute is to mediate transpositionof a gene therein, contains a region containing at least one invertedrepeat and a gene to be transposed near the repeat, and is easy to beinserted into a cell.

EXAMPLES

The present invention will be described by Examples below moreprecisely, but these Examples do not limit the present invention.

In the experiments in the present invention, eggs for injection wereobtained from zebrafish strains, Tuebingen, TL and brass and were usedfor the following experiments.

Example 1 Cloning of cDNA

Zebrafish fertilized eggs were injected with the (Tol2-tyr) plasmid and,9 hours after the injection, total RNA was extracted from 50 ofzebrafish embryos with Tri Zol Reagent (Life Technologies, Inc.) andabout 3μ g of the total RNA obtained was used for 3′ RACE and 5′ RACE,respectively.

Nested forward primers used to perform 3′ RACE are:

Tol2f2; 5′-TTGGTCAGACATGTTCATTG-3′ (SEQ ID NO:16) and Tol2f3;5′-ATGTTCATTGGTCCTTTGGA-3′, (SEQ ID NO:17) Tol2f4;5′-ATAGCTGAAGCTGCTCTGATC-3′ (SEQ ID NO:18) and Tol2f5; 5′-CTGCTCTGATCATGAAACAG-3′, (SEQ ID NO:19) Tol2f8; 5′-GCTTAATAAAGAAATATCGGCC-3′ (SEQID NO:20) and Tol2f9; 5′-AATATCGGCCTTCAAAAGTTCG-3′, (SEQ ID NO:21) andTol2f12; 5′-CTGTAATCAGAGAGTGTATGTGTA-3′ (SEQ ID NO:22) and Tol2f13;5′-ATTGTTACATTTATTGCATACAAT-3′. (SEQ ID NO:23)Nested reverse primers used for 5′ RACE are:

Tol2r4; 5′-CTCAATATGCTTCCTTAGG-3′ (SEQ ID NO:24) and Tol2r5;5′-CTTCCTTAGGTTTGATGGCG-3′. (SEQ ID NO:25)

The 3′ RACE and 5′ RACE products were gel-extracted, cloned with TOPO TACloning Kit (Invitrogen, Inc.) and sequenced using the ABI PRISM 310Genetic Analyzer.

The sequence determined is shown in SEQ ID NO:1 and the amino acidsequence of its translated region is shown in SEQ ID NO:2.

Also, the summary is shown in FIG. 1. The numbers in the parentheses arebp from the 5′ end of the Tol2 element. DDBJ/EMBL/Genbank accessionnumber for the cDNA sequence is AB032244.

Example 2 Construction of the (Tol2-tyr) Δin1ΔRV Plasmid

The (Tol2-tyr)Δin1ΔRV plasmid was first constructed by replacing theNruI-NspV of the (Tol2-tyr) plasmid with the NruI-NspV fragment of thecDNA and the resulting plasmid was digested with EcoRV and self-ligated.

Example 3 mRNA Synthesis, Injection to Embryos and PCR Analysis

The cDNA encoding the entire coding region of the transposase was clonedin pBluescript SK+ (Stratagene), linearized, digested with proteinase Kand phenol/chloroform extracted. mRNA was generated by in vitrotranscription by using T7 RNA polymerase and the mCAP mRNA Capping kit(Stratagene). The concentration and the size of the transcript wereexamined on agarose gel electrophoresis.

Zebrafish fertilized eggs were injected with 1-2 nl of a DNA solution˜25 ng/μl of a plasmid DNA) with or without the mRNA ˜5 ng/μl of theTol2mRNA) and incubated at 28° C. for ˜8 hours. Each embryo was soaked in 50μl of 10 mM EDTA, 10 mM Tris-HCl (pH 8.0), 200 μg/ml proteinase K andincubated at 50° C. for 3 hours.

Then 1 μl of the lysed embryo was used for PCR (35 cycles of 94° C. 30sec, 55° C. 30 sec and 72° C. 30 sec) using tyr-ex4f and tyr-ex5rprimers (Kawakami et al., 1998). The PCR products were analyzed on 2%agarose gel electrophoresis. The result is shown in FIG. 4.

For the DNA sequencing analysis, the PCR products were gel-extracted,cloned with TOPO TA Cloning (Invitrogen) and sequenced. The presence ofthe injected plasmid DNA in each sample was verified by PCR (25 cyclesof 94° C. 30 sec, 55° C. 30 sec and 72° C. 30 sec) using Tol2f1(5′-TCCACCCATGCTTCCAGCAGTA-3′, SEQ ID NO:28) and Tol2r3(5′-CGTTGTGGTTGCAATCCATTCAAC-3′, SEQ ID NO:29) primers.

INDUSTRIAL APPLICABILITY

The present invention results in a new protein having a transposase-likeactivity of a gene and the nucleic acid encoding the same.

Further, the present invention discloses that a transposase of adifferent family is able to generate an enzymatic activity which cancatalyze transposition of a gene in a vertebrate cell, and greatlycontributes to the development of technologies concerning thetransposition of a gene in vertebrate and the analyses of mutantsgenerated by the said transposition. On the other hand however, sincerecent gene technologies are extending from modification of a cell tomodification of an organism, a method of transposition of a gene in acell in the present invention is expected not to be limited only to themodification of a cell but also applicable to modification of thestructures and functions of genes of mammals in the medical andagricultural fields as one of the methods for modifying the organism ofthe traits. It can be expected to be a powerful method especially forthe gene therapy and the improvement of fish breeding.

1. An isolated nucleic acid encoding a protein having a transposase-likeactivity which can insert a fragment of a gene into the genome, whereinthe protein consists of the amino acid sequence of SEQ ID NO:
 2. 2. Thenucleic acid according to claim 1, wherein the nucleic acid is DNAconsisting of the nucleotide sequence of SEQ ID NO:
 1. 3. The nucleicacid according to claim 1, wherein the nucleic acid is RNA.